User:Allison K. Alix/Notebook/Thesis Research/2013/06/03

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Fluorescence Measurements and Sample Preparation for FCS Main project page
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Objectives

  • Add ThT to AuNP/DNA solution and observe Fluorescence
  • Make dilutions of AuNP/DNA/MB, DNA/MB, and MB for FCS Measurements

Procedures

Part 1: Addition of ThT to AuNP/DNA mixture

  • we want to change the volume of the AuNP/DNA mixture by only 10%

1) Add 25μL of 24.5μM ThT to 250μM AuNP/DNA (see User:Allison K. Alix/Notebook/Thesis Research/2013/05/23 Part 1)

Final concentrations of components in solution:

[AuNP] = 13.486nM

[DNA]= 1.011μM

[ThT]= 2.227μM

2) Observe fluorescence when excited at 440nM

Part 2: Preparing MB solutions for FCS

AuNP/DNA/MB solution- Prepare solutions with the following concentrations of AuNP: 1nM, 500pM, 100 pM

original concentration of AuNP in AuNP/DNA/MB solution: 9.03nM

1) 9.03nM (x) = 1nM(30μL)

x = 3.3μL AuNP/DNA/MB solution in 26.7μL PBS

[MB] = 1268nM(3.3μL)=(x)(30μL) [MB] = 140nM

[DNA] = 677.157nM(3.3μL)=(x)(30μL) [DNA] = 74.94nM

2) 1nM (x) = 500pM(30μL)

x = 15μL of 1nM AuNP/DNA/MB solution in 15μL PBS

[MB] = 140nM/2 = 70nM

[DNA] = 74.94nM/2 = 37.47nM

3) 500pM(x)=(100pM)(30μL)

x = 6μL 500pM in 24μL PBS

[MB] = 70nM/5 = 14nM

[DNA] = 37.47nM/5 = 7.5nM

DNA/MB solution- prepare solutions with the following concentrations of MB: 140nM, 70nM, and 14nM

4) 1268nM(x)=(140nM)(30μL)

x = 3.3μL DNA/MB solution in 26.7μL PBS

[DNA] = (727.5nM)(3.3μL)=(x)(30μL) [DNA] = 80.3nM

5) 140nM(x)=(70nM)(30μL)

x = 15μL 140nM DNA/MB solution in 15μL PBS

[DNA] = 80.3nM/2 = 40.15nM

6) 70nM(x)=(30μL)(14nM)

x = 6μL 70nM DNA/MB in 24μL PBS

[DNA] = 40.15nM/5 = 8.03nM

MB only solution- prepare solutions with the following concentrations of MB: 140nM, 70nM, and 14nM

7) 1268nM(x)=(140nM)(30μL)

x = 3.3μL in 26.7μL

8) 70nM = 15μL 140nM in 15μL PBS

9) 14nM = 6μL 70nM in 24μL PBS

Data

Image:Pic_of_ThTfluor05312013.png

The above graph shows the fluorescence intensity of three solutions: ThT only, ThT bound to thiol DNA, and ThT bound to thiol-DNA bound to an AuNP. This data supports the previous hypothesis that there is a decrease in fluorescence intensity as seen in the AuNP/DNA/ThT solution when compared to the DNA/ThT solution due to fluorescence quenching by the gold nanoparticle. Here we can also see that fluorescence is only observed when the ThT is in solution with DNA and not when the ThT is in solution alone. This is important as any fluorescence intensity may be attributed to the presence of DNA.

Image:Pic_of_fluor_MB.png

The above graph shows the fluorescence intensity of three solutions: MB only, MB bound to thiol DNA, and MB bound to thiol DNA bound to an AuNP. The intensity is low, but we are not having a problem with fluorescence quenching with the gold nano-particle as seen in the ThT measurements. Our next step is is run FCS measurements on these solutions at lower concentrations of AuNP (see Procedures: Part 2)





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